JPS61189228A - Production of blood coagulation factor viii - Google Patents

Abstract

PURPOSE:To obtain the titled preparation having high purity, by adding sarcosine to a solution containing blood coagulation factor VIII, and heat- treating the mixture, thereby inactivating the virus of hepatitis, etc. and removing the impurity proteins. CONSTITUTION:A blood coagulation factor VIII preparation having high purity can be produced by adding sarcosine (N-methylglycine) as a stabilizer to a solution containing blood coagulation factor VIII, and heat-treating the mixture. The hepatitis virus, etc. existing in the preparation can be inactivated without losing the activity of factor VIII, and the impurity proteins can be removed by this treatment. Similarly high-purity factor VIII can be produced by using a sugar alcohol, monosaccharide or oligosaccharide in combination with sarcosine as a stabilizer, and heat-treating the mixture. Sarcosine has high stabilizing effect to the factor VIII against heat compared with other amino acids, and is capable of effectively inactivating the viruses.

Description

Detailed Description of the Invention The present invention heat-treats a solution containing blood coagulation factor VIII in the presence of sarcosine (N-methylglycine), a highly specific stabilizer, to prevent hepatitis viruses, etc. This invention relates to a method for manufacturing highly pure preparations by inactivating pathogenic microorganisms and simultaneously removing contaminating proteins such as fibrinogen.

Blood coagulation factor VIII (hereinafter abbreviated as factor VIII) is a protein with a molecular weight of about 1 million that exists in extremely small amounts in plasma, and lack of its activity causes hemophilia A. For the treatment of bleeding tendency in hemophilia A, the most effective therapy is to replace the deficient factor (I), and the clinically most effective method for this is supplementation with concentrated preparations.

It is well known that hepatitis is transmitted by blood products. Since factor ① preparations are also contaminated with hepatitis viruses, their treatment may pose a risk to hemophilia patients. For example, commercially available factor I concentrates are manufactured from pooled plasma from several donors and have an inherent risk of hepatitis that is proportional to the number of infusions. By the way, the prevalence of HBs antibodies and HBc antibodies against hepatitis B antigen in hemophilia patients receiving treatment with these products is 8.8% each.
This is a high value of 0.90%. Solutions to hepatitis infection include producing hepatitis from plasma that is free of hepatitis viruses, developing a vaccine against the virus and administering it at the time of the first dose of factor Ⅰ preparation, and performing heat virus inactivation treatment. However, the virus identification method used by 1ii72 is not perfect, and it is medically problematic to use a vaccine that does not play a sufficient role in an emergency, and at this stage it is incomplete as a preventive method. be. Until now, in plasma-derived products other than coagulation factors, such as albumin and antithrombin III, the spread of hepatitis has been prevented by heating an aqueous solution containing these proteins at 60°C for 10 hours in the presence of a specific stabilizer. It had been(
Grellis, S et al. J, C11n, Inve
st,, vol 27+ P, 239-245 (194
B) and Tabor, E et al. Thrombosis
Re5archvol 22. P233-238
(1981)). That is, a highly specific stabilizer is added to the plasma protein solution to inactivate the infectivity of the hepatitis virus while keeping the plasma protein stable. In recent years, attempts have been made to similarly heat factor Ⅰ concentrates in solution. Heimburger et al. heated purified factor 1 in a sucrose-glycine solution at 60°C for 10 hours. As a result, the activity of factor ① was preserved during the heat treatment, but the recovery rate was as low as about 8%. In contrast, 1" Blood coagulation factors and their manufacturing method (Japanese Unexamined Patent Publication No. 55-14
No. 5615)-1 and [Heat treatment method for blood coagulation factors (
JP-A-59-134730) I has been submitted. In the former, the sucrose concentration was set to 50%, and 2M glycine was added to this to increase the recovery rate to 60% after heating at 60°C for 10 hours]
- Raised. In the latter case, sucrose and sorbitol were added as stabilizers at an even higher concentration of 1.5 g per ml of the factor (I) solution, increasing the recovery rate in -1- to 57-58%. However, it has been reported that in the presence of high concentrations of sugar, viruses are also stabilized against heat (Miyamoto et al., 4th [1] Abstracts of the Society on Thrombosis and Hemostasis, p. 6).
1 (1984)). Considering the above, the currently available heat treatment technology stabilizes factor II and achieves high yield by adding high concentrations of sugars.
On the other hand, since this condition simultaneously stabilizes the virus, it is considered to be insufficient as a method for inactivating hepatitis viruses.

The present inventors have found that sarcosine is effective as a stabilizer during the heat treatment of factor (I) in order to improve these insufficiencies. Table 1 shows the changes in activity when purified factor ① was heated at 60°C in the presence of various amino acids. The amount of amino acid added was a final concentration of 0.2M, and the pH was 70. As is clear from the results in Table 1, sarcosine has an extremely high stabilizing effect compared to other amino acids.

Table (2) Various concentrations of sarcosine were added and
The recovery rate of factor ① when heated for a certain period of time is shown. As shown in the table, the stabilizing effect is proportional to the concentration of sarcosine, but sarcosine is easily soluble in water and can maintain a high concentration. In addition, although sarcosine is effective as a stabilizer in 14 days, as shown in Table 3, sarcosine can also be used in combination with sugars.
Similarly, a high stabilizing effect can be obtained. In this case, a sugar concentration as low as about 10% is sufficient. Furthermore, by removing the precipitate produced by the heat treatment by centrifugation, highly pure factor ① can be obtained. The present invention was completed based on the above new findings.

Table 1 Stabilizing effect of various amino acids on factor Ⅰ activity upon heating at 60°C Stabilizers k -min-' Sarcosine 0.0335 Glycine
0185 Alanine
0128 Hydroxyproline 02764-amino-n
-butyric acid 01462-amino-jso-butyric acid
0246 Table: Stabilizing effect of factor Ⅰ activity by addition of sarcosine upon heating at 260°C 6.2 100 785.3
100 512, 5 100
9.51.3 100 0 0.6 100 0 Table 3 Stabilizing effect of factor Ⅰ activity upon heating at 60°C by addition of sarcosine and 10% (W, /V ) sorbitol 5.4 10 100 714.2
10 100 522.1
10 100 15.61.0 10
100 2.8 In the present invention, sarcosine is added to a solution containing factor Ⅰ at a final concentration of 1 to 8 M and heated at 50 to 90°C.
By heating for 0.5 to 30 hours, the infectivity of pathogenic microorganisms such as hepatitis viruses is inactivated, and contaminant proteins such as fibrinogen are removed. Alternatively, in addition to sarcosine, a stabilizer selected from monosaccharides, oligosaccharides, and sugar alcohols, such as sucrose, sorbitol, galactose, and maltose, is used in combination and the same treatment is performed to obtain highly purified factor get.

The raw materials used in the present invention are CPD,
Fresh plasma collected and separated in the presence of an anticoagulant such as ACD, or fresh plasma obtained by apheresis, cryoprecipitate obtained by freezing and thawing these, or further combined with aluminum hydroxide gel. Fractions purified using ion exchange resins, control topore glass beads, etc. can be considered, but there are no particular limitations as long as the fractions contain human-derived factor ①. This is because the stabilizing effect of sarcosine is almost the same for factor (I) of any degree of purification, so it may be added at any stage during the manufacturing process of factor (I) preparations. The concentration of Factor (1) in the Factor (1) aqueous solution used after heat treatment is 001 to 100 units/ml, preferably 1 unit/7 or more.

The protein content is preferably 10/- or less.

For samples containing a higher concentration than this, it is preferable to dilute the sample with physiological saline or the like and then perform the heat treatment. The pH of the solution is generally 60-9.0, preferably 70-7.
．． 8 is desirable.

The heat-treated factor ① solution was cooled to 10-15°C, and then 1. Centrifuge and remove the precipitated fraction. Stabilizers such as sarcosine contained in Nisumi are removed by methods such as ultrafiltration and gel filtration. The obtained aqueous solution containing factor (I) is further purified, if necessary, by column chromatography, fractional precipitation, adsorption chromatography, etc., or concentrated by ultrafiltration, and dispensed into glass vials.
Freeze-dry to obtain a factor Ⅰ concentrated preparation.

The present invention will be explained below using examples.

Note that the present invention is not limited to these examples.

Example 1 An anticoagulant such as ACD or CPD was added to fresh human blood and centrifuged to precipitate blood cell components to obtain plasma. This was frozen in a -60°C freezer, then thawed in a 2°C water bath, and the resulting precipitate fraction (cryoprecipitate) was collected by centrifugation. The cryoprecipitate 1~ collected in this way was mixed with 101 5M sarcosine (pH 7,
0) and heated at 60°C for 10 hours. Thereafter, the solution was cooled to 15° C., the precipitate was removed by centrifugation, and the supernatant was collected. It was loaded onto a Sephadex G-25 column equilibrated with physiological saline containing 0.296 sodium citrate, and the stabilizer was removed. It was concentrated by ultrafiltration, dispensed into glass vials, and lyophilized to obtain a factor (2) preparation.

The recovery rate of factor (1) during this operation was 12%, and 61% before and after the heating operation. The fibrinogen content in the obtained product was 0.1 mg or less per unit of factor 1.

Example 2 Human plasma containing ACD as an anticoagulant obtained by apheresis plasma collection was frozen in dry ice methanol and then dissolved in a refrigerator at 4°C. The resulting cryoprecipitate fraction was centrifuged. It was collected. This fraction I Kg is 101! It was dissolved in a 2.5M sarcosine solution (pH 7.3) containing 10% sorbitol and heated at 60°C for 10 hours. After cooling the solution to 0°C, it was centrifuged and one supernatant was collected. Sephadex G-25 equilibrated with physiological saline containing 0.2% sodium citrate.
Loaded onto a column, removed stabilizer and concentrated by ultrafiltration. This was dispensed into glass vials and freeze-dried to obtain a preparation exhibiting properties substantially similar to those of Example 1. The recovery rate for factor Ⅰ was 11%. Before and after heating, 58% of factor Ⅰ activity was recovered.

Example 3 Cryoprecipitate was obtained from human plasma using the method shown in Example 1.
1 was taken and heat-treated in the presence of 5M sarcosine. 400 ml of Al(
After adding the gel and stirring gently for 1 hour, the supernatant was collected by centrifugation. In addition to this, Control Topore Glass Beads 51 were washed in advance with physiological saline containing 02% sodium citrate! was added and stirred. The mixture was placed on a glass filter to separate the gel and the eluent was collected.

The gel was washed with 10% physiological saline, and the eluate was fractionated and mixed with the previously fractionated solution. pH 0.1. NHC1
According to 6.6, DL: AE-Sephadex A-50
Load the column (Bed, Vol, 5/) with 0.1
It was eluted with a gradient of 5 to 2.0 M NaCl, and fraction 41 containing factor Ⅰ activity was collected. The product was desalted and concentrated by ultrafiltration, dispensed into glass vials, and freeze-dried to obtain a product. The obtained product contained almost no fibrinogen, coagulation factors such as Nos. 1, 2, IX, X, and M.

Example 4 Cryoprecipitate was recovered from plasma using the method of Example 1. 0.1 containing 20mM sodium citrate
Dissolved in M sodium chloride (PH7.0). overall 3
% volume of AI(OH) was added, stirred, and centrifuged to obtain a supernatant. Add PEG4.000 to this supernatant at a final concentration of 3% (W/V
), stirred gently for 2 hours, and removed the precipitate fraction by centrifugation. Furthermore, PEG4,00 was added to the obtained supernatant.
0 to a final concentration of 5.5% (W/V), and
H was adjusted to 6.0 with citric acid solution. Centrifuge and
A precipitate fraction rich in factors was obtained. This factor ■ enriched fraction
Dissolved in M sarcosine solution (pH 7,2) and incubated at 60°C for 1
Heat treatment was performed for 0 hours. Sarcosine was removed by gel filtration, the concentration was adjusted by ultrafiltration, 0.3% sodium citrate was added, the mixture was dispensed into glass vials, and lyophilized to produce a concentrated factor II preparation. No fibrinogen was detected in the preparation, and the recovery rate of factor Ⅰ was about 10% throughout the entire process.

Example 5 A lyophilized product of a commercially available factor Ⅰ concentrate preparation (250 units ρf) was dissolved in 3M sarcosine (pH 7.3) containing 8% sucrose to give 20 units/m12, 60
After heating at ℃ for 10 hours, the clear liquid was collected by centrifugation, and after removing the stabilizer by ultrafiltration, it was concentrated to 2 times, and sodium citrate (1.8 rq/ml) and sodium chloride (0.0 , 3 mg/mp) was added, dispensed into glass vials, and freeze-dried. The recovery rate for factor ■ was 49%.
Met.

Claims (1)

[Claims] 1. In the manufacturing process of blood coagulation factor VIII preparations, by adding sarcosine (N-methylglycine) as a stabilizer to a solution containing blood coagulation factor VIII and subjecting it to heat treatment, Blood coagulation factor VIII, which is characterized by inactivating viruses such as hepatitis contained in the preparation without losing the activity of factor VIII, and removing contaminating proteins to produce highly pure preparations. Method for manufacturing factor preparations. 2. By using sugar alcohol, monosaccharide or oligosaccharide in combination with sarcosine as a stabilizer and applying heat treatment, viruses such as hepatitis contained in the preparation can be removed without losing the activity of factor VIII. A method for producing a blood coagulation factor VIII preparation, which is characterized by inactivating the blood coagulation factor VIII and removing contaminating proteins to produce a highly pure preparation.